The present study identified antibody specificities associated with neutralization breadth. We found that both anti-MPER and anti-CD4i antibody titers were correlated with the number of viruses neutralized. Furthermore, antibodies to CL were found to be correlated with neutralization breadth and with anti-MPER antibodies. We did not find anti-gp120 or anti-CD4bs antibodies more frequently in samples with breadth. However, a number of samples had neutralizing antibodies that bound monomeric gp120, some of which were shown to mediate cross-neutralization.
For this study, we used samples collected from South African HIV-1-infected blood donors who were assumed to be infected with HIV-1 subtype C, since this is the predominant subtype in the country. Indeed, gp160 sequence analyses of 15 samples confirmed these to be HIV-1 subtype C (not shown). After exclusion of the incident infections, we found 16 out of 70 (23%) plasmas from chronically HIV-1-infected individuals to have BCN antibodies. However, this frequency may be confounded by the fact that these samples were obtained from blood bank donors and therefore may be enriched for healthy individuals who were unaware of their HIV infections. While it is not clear that disease progression is related to the presence or lack of neutralizing antibodies, it is fair to presume that a healthy immune system may be more likely to generate these antibodies. Nevertheless, our data are in line with other recent studies that report similarly high proportions of chronically infected individuals with antibodies able to mediate cross-neutralization (11
Fourteen of the 16 BCN samples included here have been previously investigated (1
). Our study builds on this by including non-BCN samples to ascertain which specificities were associated with breadth. In addition, we used the subtype C strain Du151 for both adsorption and neutralization experiments to maximize the opportunities for detecting relevant antibodies. Similar to the Binley study, we found antibodies targeting the CD4bs using gp120-adsorbed antibody preparations. However, these antibodies were found in plasmas with a range of neutralizing activities. They mediated gp120 binding, as well as neutralization of the laboratory-adapted strain HxB2, but had limited ability to neutralize the primary virus, Du151. In one sample (BB10), CD4bs antibodies were shown to mediate heterologous neutralization. However, these antibodies were not BCN, since this plasma inhibited only 40% of the panel. While recent studies have identified rare individuals with broadly neutralizing anti-CD4bs antibodies, in most cases anti-CD4bs antibodies have been shown to have only limited breadth, similar to what we have described (1
). This is analogous to the reactivities of existing CD4bs MAbs, such as b6, b13, 15e, and F105, which have little activity against primary isolates (3
). Overall, our data suggest that while anti-CD4bs antibodies are readily produced during HIV-1 infection, they vary both qualitatively and quantitatively among different polyclonal plasma samples.
In our study, monomeric Du151 gp120 was able to adsorb >50% of neutralizing antibodies against the Du151 virus in 7/16 (44%) plasmas tested. In two cases (BB55 and BB70), these anti-gp120 antibodies were shown to be responsible for the heterologous neutralizing activity. The finding that a significant number of BCN plasmas can be adsorbed with monomeric gp120 may be somewhat surprising given the prevailing notion that neutralizing antibodies bind trimeric proteins (34
). It is perhaps also intriguing that monomeric gp120 vaccines have been unable to induce equivalent antibodies in vivo, given that the epitopes are at hand. It suggests either that these antibodies are not produced at sufficiently high titer in vaccinees to mediate neutralization or that these epitopes cannot be induced by a protein mimic, similar to the failed attempts to induce neutralizing antibodies to other HIV epitopes (25
). Another possibility is that BCN antibodies require repeated antigenic stimulation for an extended period, similar to natural HIV infection.
Interestingly, we found a good association between neutralization breadth and the presence of anti-CD4i and anti-MPER antibodies. CD4i antibodies develop in natural HIV-1 infection (8
), although their role in virus neutralization remains unclear given the poor accessibility of the site (24
). Nevertheless, CD4i antibodies have been found in samples with neutralization breadth (30
), in support of what we found here. Moreover, our data suggested that anti-CD4i antibodies in BB55 contributed to heterologous neutralization, although its epitope may overlap with the CD4bs, as was evident from the data using the D368R/E370A mutant. Further studies to assess the contribution of anti-CD4i antibodies to virus neutralization may shed light on new targets for vaccine design.
A number of groups have recently shown that anti-MPER antibodies are found in samples with neutralization breadth that in some cases were identified as 4E10-like, Z13-like (1
), or 2F5-like (50
). While we also found anti-MPER antibodies, they did not appear to correspond to either 4E10 or 2F5 specificities. Based on the chimeric mapping data of six samples, we speculate that we have at least three distinct specificities targeting the MPER. BB25 and BB47 may contain Z13-like antibodies based on their neutralization of C4. BB68 and BB70 showed a similar pattern of neutralization that was distinct from that of BB28 and BB34. Antibodies in BB34 have been extensively analyzed and shown to map to a novel epitope (E. S. Gray, unpublished data). These data suggest that the MPER is immunogenic and is recognized in diverse manners by the immune system. While these observations suggest that both anti-MPER and anti-CD4i antibodies may be involved in the neutralization breadth displayed by these samples this remains to be fully demonstrated, as it is possible that these antibodies are simply a surrogate for other specificities that confer cross-neutralization.
HIV infection is associated with hypergammaglobulinemia and with elevated serum titers to a variety of self-antigens, including anti-phospholipids, anti-nuclear antibodies, and RF (4
). Here, we found anti-CL antibodies in 36% of the plasma samples tested, consistent with reports by others (48
). While anti-CL antibodies are frequently found in HIV-1 infection, they are not significantly associated with thrombotic events or other manifestations of anti-phospholipid syndrome (4
). In this study, we found anti-CL antibodies to be significantly associated with neutralization breadth. In addition, we found that anti-CL antibodies were correlated with anti-MPER titers. Similar observations have been reported recently by another group (31
), although they measured anti-MPER peptide-binding antibodies in sera while we measured anti-MPER neutralization titers. Nevertheless, these results strongly suggest that anti-MPER and anti-CL antibodies are linked and related to the development of broad neutralization. However, our results do not explain if this autoreactivity is a property of the same antibody mediating neutralization, as has been suggested by MAbs 4E10 and 2F5 (17
). Alternatively, these autoantibodies represent a parallel humoral response, perhaps indicative of a particular genetic background that supports the elicitation of neutralizing antibodies. Thus, it will be of interest to determine if these anti-CL antibodies cross-react with HIV proteins or, even more importantly, if they have neutralizing activity.
Overall, our data extend recent studies examining the antibody specificities associated with neutralization breadth and suggest additional insights into their properties. While antibodies targeting specific sites could be identified in some cases, the majority are still unknown. It is likely that cross-reactive antibodies target epitopes on the trimeric structure of the functional envelope glycoprotein or on gp41. Although rare cases of cross-neutralization due to a single antibody specificity, such as anti-CD4bs or anti-MPER, have been reported (30
), it is highly likely that more than one antibody confers neutralization breadth. In support of this, we and others have shown that multiple known specificities can be found in an individual plasma sample (such as BB70, which contained anti-gp120 and anti-MPER antibodies). A recent study examining the repertoire of anti-gp140 antibodies did not find a single specificity that accounted for the neutralization breadth exhibited by the plasma, suggesting that multiple antibodies with more limited activities contributed to breadth (45
). Major questions that remain are how neutralization breadth develops and why it develops only in some individuals (are host genetic factors involved?). Longitudinal studies are needed in which the evolution of various antibody specificities is dissected and their contributions to overall neutralization breadth are determined. The identification of the precise epitopes of these BCN antibodies remains a top priority for vaccine research.